An in vitro Model of Schlemm’s Canal Endothelium Reveals Pressure Dependent Formation of Giant Vacuole-Like Structures

Abstract

Purpose: : Transport across Schlemm’s canal endothelium (SCE) and SCE deformation during giant vacuole and pore formation are important for the generation of outflow resistance. In this project, we investigate the pressure dependence of endothelial deformation and formation of "giant vacuole-like" structures in an in vitro endothelial perfusion model that mimics the in vivo mechanical environment of SCE.

Methods: : Our existing perfusion/microscopy system (ARVO 2007 #2073) allows dynamic visualization of endothelial cells during basal-to-apical directed perfusion. Human umbilical vein endothelial cells (HUVECs) or human SCE cells were seeded at confluence (10⁵ cells/cm²) on track-etched (0.4 µm) polyester membranes and cultured for at least 2 days to reach 8.3±2.0 Ω cm². For visualization, cells were stained with cytoplasmic and nuclear vital dyes, and placed within the perfusion/microscopy apparatus. Six HUVEC and 3 SCE monolayers were perfused at a defined basal-to-apical directed flow rate (2-30 µL/min), while epifluorescence videomicroscopy was used to image cytoplasmic and nuclear deformation.

Results: : At the onset of flow, micron-sized circular void regions appeared within an otherwise uniformly stained cytoplasm, which we interpret as focal sites of cell-substrate detachment and apical cell displacement into thin domes or "giant vacuole-like" structures (GVLs). At low pressure (1-2 mmHg), GVLs were small (2-4 µm) and appeared scattered under cells and along cell margins, occasionally forming racemose clusters. At higher pressure (5-7 mmHg), GVLs were larger (10-15 µm) and were associated with significant cytoplasmic and nuclear deformation. Larger GVLs expanded viscoelastically from the onset of flow and recoiled after flow cessation, without loss of vital dye indicating preserved cell viability.

Conclusions: : Giant vacuole-like structures appeared during basal-to-apical directed perfusion in both HUVEC and SCE monolayers, exhibiting pressure-dependence and viscoelastic behavior. These data suggest that giant vacuole formation may not be a unique property of SCE cells, and may be studied in vitro using a perfusion/microscopy system.Acknowledgements: We thank Dr. Dan Stamer (U. of Arizona) for providing SCE cells.

Keywords: outflow: trabecular meshwork • intraocular pressure